Regulation of the biochemical function of motif VI of HCV NTPase/helicase by the conserved Phe-loop.
In previous works, we demonstrated a potent inhibition of diverse protein kinase C (PKC) functions by a fragment of nonstructural protein 3 (NS3) of hepatitis C virus (HCV), mainly mediated by the Arg-rich amino acid motif HCV(1487-1500). This sequence is localized on the surface of Domain 2 of the NS3 NTPase/helicase in direct vicinity to a flexible loop that is localized between Val1458 and Thr1476. Here, we assessed the regulation of the accessibility of the Arg-rich amino acid motif for PKC by this loop, using two variants of domain 2. The first construct, termed NS3d2Delta, comprises the complete domain, HCV(1361-1503), devoid the loop. The second variant, NS3d2wt corresponds to wild type domain 2. The results indicated an enhanced inhibitory potential of NS3d2Delta towards rat brain PKC and towards the majority of PKC isoforms. This effect and the accompanying change of the mode of inhibition from a mixed mode, exerted by NS3d2wt to a competitive mode, exerted by NS3d2Delta are caused by the deletion of the loop. Accordingly, the presence of the intact loop abolished the binding of domain 2 to the tailed duplex RNA used as helicase substrate, without affecting the binding of dsDNA. Furthermore, a direct competition of dsRNA and PKC for the same binding site HCV(1487-1500), could be documented. The binding of dsRNA to NS3d2Delta previously overlaid with PPKCalpha was reduced to 30% and completely abolished in case of NS3d2Delta overlaid with cAMP-dependent protein kinase A (PKA).
Hartjen
Philip
P
Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
Medom
Berthe Kamdem
BK
Reinholz
Michael
M
Borowski
Peter
P
Baier
Andrea
A
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D043583:Nucleoside-Triphosphatase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D043583:Nucleoside-Triphosphatase; D020033:Protein Isoforms; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D020365:RNA Helicases; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases